JP4164129B2 - Bacterial antigen-specific nucleic acid molecules and uses thereof - Google Patents

Abstract

The present invention relates to nucleic acid molecules derived from; a gene encoding a transferase; or a gene encoding an enzyme for the transport or processing of a a polysaccharide or oligosaccharide unit, including a wzx gene or a wzy gene, or a gene with a similar function; the gene being involved in the synthesis of a particular bacterial polysaccharide antigen, wherein the sequence of the nucleic acid molecule is specific to the particular bacterial polysaccharide antigen. Polysaccharides to which the invention relates include O antigens. The invention also relates to methods of testing samples for the presence of one or more bacterial polysaccharide antigens, using the nucleic acid molecules of the invention, and to kits containing the nucleic acid molecules of the invention.

Description

配列表
（１）一般情報
（ｉ）出願人：リーブス，ピーター アール ワン，リー
（ii）発明の名称：バクテリア抗原特異核酸分子およびその用途
（iii）配列の数：４
（iv）住所
(A) 宛て先：トーマス グムレイ
(B) 街：168ウォカーストリート
(C) 市：ノースシドニー
(D) 州：ニューサウスウェールズ
(E) 国：オーストラリア
(F) 郵便番号：2068
（ｖ）コンピュータ読取形態
(A) 媒体：フロッピーディスク
(B) コンピュータ：IBM PC互換機
(C) オペレーション・システム：PC-DOS/MS-DOS
(D) ソフトウェア：パテントイン リリース#1.0,バージョン#1.30
（vi）出願データ
(A) 出願番号
(B) 出願日
(C) 分類
（viii）代理人
(A) 氏名:グムレイ，トーマス ピー
（ix）通信情報
(A) 電話番号：99575944
(B) FAX番号：99576288
（２）配列番号１の情報
（ｉ）配列の特徴
(A) 配列の長さ：14516塩基対
(B) 配列の型：核酸
(C) 鎖の数：一本鎖
(D) トポロジー：直鎖状
（ii）配列の種類：DNA
（iii）ハイポセチカル配列：NO
（iv）アンチセンス：YES
（ｖ）起源：
(A) 生物名：大腸菌
（ix）配列：配列番号１

（２）配列番号２の情報
（ｉ）配列の特徴
(A) 配列の長さ：14024塩基対
(B) 配列の型：核酸
(C) 鎖の数：二本鎖
(D) トポロジー：直鎖状
（ii）配列の種類：DNA
（iii）ハイポセチカル配列：NO
（iv）アンチセンス：YES
（ｖ）起源：
(A) 生物名：大腸菌
（vi）注 最初の19塩基対はロングPCRに使用したプライマーからである。
（ix）配列：配列番号２

（２）配列番号３の情報
（ｉ）配列の特徴
(A) 配列の長さ：12441塩基対
(B) 配列の型：核酸
(C) 鎖の数：二本鎖
(D) トポロジー：直鎖状
（ii）配列の種類：DNA（ゲノミック）
（iv）アンチセンス：YES
（ｖ）起源：
(A) 生物名：サルモネラ・エンテリカ血液型亜型ミュエンヒェン血清グループＣC2
（ix）配列：配列番号３

（２）配列番号４の情報
（ｉ）配列の特徴
(A) 配列の長さ：22080塩基対
(B) 配列の型：核酸
(C) 鎖の数：二本鎖
(D) トポロジー：直鎖状
（ii）配列の種類：DNA（ゲノミック）
（iv）アンチセンス：YES
（ｖ）起源：
(A) 生物名：サルモネラ・エンテリカ血液型亜型ティフィリウム（血清グループB）
（ix）配列：配列番号４

Technical field
The present invention relates to novel nucleotide sequences located in gene clusters that control the synthesis of bacterial polysaccharide antigens, particularly O antigens, as well as the detection of bacteria expressing specific polysaccharide antigens (especially O antigens) and polysaccharide antigens of these bacteria (especially O antigens). Relates to the use of these nucleotide sequences to identify
Background art
It is well known that enteropathogenic E. coli strains are responsible for human diarrhea and hemorrhagic colitis and are potentially life threatening sequelae including hemolytic uremic syndrome and thrombotic thrombocytopenic purpura Lead to. Some of these strains are commonly found in livestock, and human infections are usually the result of consumption of improperly processed and contaminated meat or dairy products. It is known that the O-specific polysaccharide component ("O antigen") of lipopolysaccharide is a major virulence factor of enteropathogenic E. coli strains.
The Escherichia coli O antigen is highly polymorphic, and 166 different shapes have been identified for this antigen; Ewing, WH [Edwards and Ewings “Identification of Enterobacteriaceae” Elsbyer, Amsterdam (1986)] examines 128 different O antigens, but Lior H. (1994) has expanded its number to 166 ["Coliform classification", domestic animals and E. coli in humans, 31-72, edited by CL Gyles, CAB International].Salmonella entericaThe genus has 46 known O antigen types [Popoff, M. Y. et al. (1992) "Salmonella entericaAntigen shape of serovars "6th edition,Salmonella entericaWHO Collaborating Center for Reference and Research, Pasteur Institute, Paris France].
An important step in determining the biosynthesis of the O antigen and thus the mechanism of polymorphism is to characterize the gene cluster that controls O antigen biosynthesis. Genes specific for O antigen synthesis are generallyE.coli In the gene cluster of 45 minutes on the chromosomal map of K-12 [Backmann, B. J. 1990 “Linkage map of Escherichia coli K-12”, Microbiol. Rev. 54: 130-197],Salmonella enterica It is located in the corresponding position in LT2 [Sanderson et al. (1995), “Salmonella enterica typhimurium gene map ", VIII edition, Microbiol. Rev. 59: 241-2303]. In both cases, the O antigen gene clusterE.coliandS.entericaAs close to the gnd gene as in the strain [Reeves PR (1994) “Lipopolysaccharide biosynthesis and assembly” 281-214, A. Neuberger and LLM van Deenen Compilation “Bacterial Cell Membrane, New Integrated Biochemistry”, Volume 27, published by Elsbyer Science. These genes encode enzymes for the synthesis of nucleotide diphosphate sugars and for assembling the sugars into oligosaccharide units, and generally for the polymerization to O antigens.
E.coli O gene cluster is extensiveE.coli Although cloned for the O antigen, the O antigens of O7, O9, O16 and O111 have been studied in more detail, and O9 and O16 have been fully characterized to date with respect to nucleotide sequences [Kido, Turkishf. , Sugiama, Uchiya, Sugihara, Komatsu, Kato and Yang (Kido N, Torgov VI, Sugiyama T., Uchiya K., Sugihara H., Komatsu T., Kato N. & Jann K.) (1995) “Escherichia coli O9 polysaccharide expression:E.coli Sequencing of the O9rfb gene cluster, characterization of mannosyltransferase, and proof of ATP-binding cassette transport system ", J. of Bacteriol. 177, 2178-2187; Stevenson, Neil, Riu, Hobbs, Packer, Batley, Redmond, Lindgeist , And Reeves (Stevenson G., Neal B., Liu D., Hobbs M., Packer NH, Batley M., Redmond JW, Lindguist L. & Reeves PR) (1994) "E.coli Construction of K-2 O antigen and sequence of its rfb gene cluster ", J. of Bacteriol., 176, 4144-4156; Jayaratne, P. et al. (1991)" Escherichia coli O9: GDP-mannose formation in K30 Cloning and analysis of overlapping rfbM and rfbK genes involved in and the involvement of the rfb gene in group 1K 30 capsular polysaccharide synthesis ", J. Bacteriol. 176: 3126-3139; Valvano, MA and Crosa, JH ( 1989) “Human invasionE.coli O: of the chromosomal gene that determines the O7 lipopolysaccharide antigen of the K1 strainE.coli Molecular cloning and expression in K-12 ", Inf and Immun. 57: 937-943; Marolda, C. L. and Valvano, M.A. (1993);" VW187 strain (E.coli Identification, expression and DNA sequence of GDP-mannose biosynthetic genes encoded by the O7rfb gene cluster of O7: K1) ", J. Bacteriol. 175: 148-158].
Bastin, D. A. et al. (1991) ["E.coli Of the rfb gene cluster that determines the O antigen of the O11 strainE.coli Molecular cloning and expression in K-12 ",^{Mol Microbiol}5: 9, 22223-22231] and Bastin, D.A. and Reeves, P. R. (1995) ["E.coliSequence and analysis of O111 O antigen gene (rfb) cluster "^Gene164: 17-233]E.coli Separate the chromosomal DNA encoding the O111rfb region,E.coli Six open reading frames (orfs) characterizing the 6992 bp fragment of O 111rfb have been identified in the 6962 bp partial fragment, and the linear sequence of these orfs is the GDP-mannose pathway genes, rfbK and rfbM, and other rfb and cps. It was revealed that it is homologous with the gene.
Salmonella entericaThe nucleotide sequences of loci that control O antigen expression of B, A, D1, D2, D3, C1, C2 and E have been characterized [Brown, P. K., L. K. Romana and P. R. Reeves ((1991) "Group C2S.entericaCloning of rfb gene clusters from: comparison with rfb regions of groups B and D, Mol. Microbiol. 5: 1873-1881; Jiang, XM, B. Neal, F. Santiago, SJ Lee, LK Romana, and PR Reeves ) (1991)Salmonella entericaStructure and sequence of rfb (O antigen) gene cluster of subtype typhimurium (LT2) ", Mol. Microbiol. 5: 692-713; SJ Lee, LK Romana, and PR Reeves (1992) ) "Group C1S.entericSequence and structural analysis of the rfb (O antigen) gene cluster from strain a ", J. Gen. Microbiol. 138: 1843-1855; Louis, Berma, Romana, and Reeves (Lui, D., NK Verma, LK Romana, and PR Reeves) (1991)Salmonella entericaRelationship between rfb regions of blood type subtypes A, B and D ", J. Bacteriol. 173: 4814-4819; Verma, N. K. and P. Reeves (1989)" Group A and Group DSalmonella entericaIdentification and sequence of rfbS and rfbE to determine the antigen specificity of ", J. Bacteriol. 171: 5694-5701; Wang, L., L. K. Romana, and P. R. Reeves (1992)"Salmonella enterica Molecular analysis of entericae group E1rfb gene cluster: genetic basis of O antigen and major polymorphisms ”, Genetics 130: 429-443; Wyk, P. and P. Reeves (1989)“ Group BSalmonella entericaeIdentification and sequence of an abequos synthase that confers antigen specificity to the gene: homology to galactose epimerase ", J. Bacteriol. 171: 5687-5633; Tian, Hobbs, and Reeves (Xiang, SH, M. Hobbs , and PR Reeves) 1994, “Group D2S.entericaMolecular analysis of strain rfb gene cluster: Evidence of its origin from an insertion sequence mediated recombination event between group E and D1 strains ", J. Bacteriol. 176: 4357-4365; Curd, Riu and Reeves (Curd, H., D. Liu and PR Reeves)Salmonella entericaRelationship between group B, D1, D2 and D3O antigens ", J. Bacteriol. 180: 1002-1007].
Closely relatedShigella (actuallyE.coliCan be considered part ofS. dysenteriaeandS.flexneriThe O antigen of is completely sequenced and is flanked by gnd [Klena JD & Schnaitman CA (1993) “Functions of the rfb gene cluster andShigella dysenteriaeRfe gene in the synthesis of O antigen by 1 ", Mol. Microbiol. 9: 393-402: Morona, Mabris, Fararino and Manning (Morona, R., Mavris, M., Fallarino, A. & Manning, P.) ( 1994)Shigella flexneriCharacterization of the rfc region ", J. Bacteriol. 176: 733-747].
O antigens of enteropathogenic E. coli strains andS.entericAs long as the O antigen of a strain is the main virulence factor and is highly polymorphic,E.coliDetectS.entericaThere is a real need to develop highly specific, sensitive, rapid, and inexpensive diagnostic assays for detecting. Also,E.coliIdentifying the O antigen of the strain,S.entericaThere is a real need to develop diagnostic assays to identify strain O antigens.E.coliThese requirements for the detection of EHEC (enteric pathogenic hemorrhagicE.coli) Stocks, but this is the most necessary area.E.coliIn ETEC (enteric toxicityE.coliI'm interested in diagnosis.
The first diagnostic system adopted in this areaE.coli O antigen expression strain orS.enterica  A large amount of antiserum prepared against the O antigen expression strain was used. This technique is inherently difficult with respect to the preparation, storage and use of its reagents, and the time required to obtain meaningful diagnostic results.
S.entericaThe nucleotide sequence derived from the O antigen gene cluster of the strain isS.entericaIt has been used to determine O antigen [Luk, J.M.C. et al. (1993) “S.entericaSelective amplification of the Abequoose and Palatose synthase genes (rfb) by polymerase chain reaction for identification of major serogroups (A, B, C2, and D) ", J. Clin. Microbiol. 31: 2118-2123]. All rfb of subtypes Typhimurium, Paratyphi A, Typhi, Muenchen and Anatum representing groups B, A, D1, C2 and E1, respectively. The complete nucleotide sequence characterization ahead of the locus allowed Luke et al. To select oligonucleotide primers specific for these serogroups. Thus, Luke's method isS.entericaNucleotides observed to identify serotype-specific oligonucleotides by arranging known nucleotide sequences corresponding to CDP-adequose and CDP-palatose synthesis genes within the O antigen region of serogroups E1, D1, A, B and C2. Based on using sequence differences.
In an attempt to determine the O antigen serotype of an Escherichia coli strain producing Shiga toxin-like toxin, Paton, AW et al. (1996) ["hemolytic uremic disease caused by dried fermented sausage contaminated with Escherichia coli producing Shiga toxin-like toxin" Molecular microbiological study of syndrome development ", J. Clin. Microbiol. 34: 162-1627] used oligonucleotides derived from the wbdI (orf6) region,E.coli  In PCR diagnostic assays believed to be specific for O111E.coli  Derived from the O111 sequence. According to an unpublished paper, the Patong et al. Method is flawed in that the nucleotide sequence derived from wbdI may not specifically identify the O111 antigen, and in fact the detection is a false positive result. . Patong et al. Have detected five O111 antigen isolates by PCR and have revealed that only three of these isolates have detected bacteria that actually react with O111-specific antisera.
Disclosure of the invention
Although we do not want to be bound by a specific hypothesis, we now believe that the false positives seen by the Patong et al. Method are that the nucleic acid molecule employed by the Patong et al. Genes with functions [Bastin DA and Reeves, PR (1995),E.coliThis is due to the fact that it is derived from the sequence and analysis of the O antigen gene (rfb) cluster of O111, Gene, 164: 17-23]E.coliThey also believe that they lack the nucleotide sequence specificity necessary to identify the O antigen. The present inventors currently believe thatS.entericaOr many of the nucleic acid molecules derived from sugar pathway genes expressed in other enteric bacteria also appear to lack the nucleotide sequence specificity necessary to identify specific O antigens or specific serotypes It is.
In this regard, the genes for polysaccharide antigen synthesis include those related to the synthesis of sugars present in the antigen (sugar pathway genes) and those related to the handling of these sugars that form polysaccharides. It is important to keep in mind. The present invention is primarily concerned with the latter group of genes, particularly assembly and transport genes such as transferase, polymerase and flippase genes.
The inventors surprisingly found that using nucleic acid molecules derived from specific assembly and transport genes within the O antigen gene cluster, particularly transferase, wzx and wzy genes, the specificity of O antigen detection and identification. It has been found that it can be improved. The present inventors are not necessarily limited to the detection of specific O antigens encoded by the nucleic acid molecules exemplified herein, but are widely applicable to the detection of bacteria expressing O antigens and the identification of general O antigens. I believe it is applicable. Furthermore, because of the similarity between gene clusters involved in the synthesis of O antigen and those of other polymorphic polysaccharide antigens such as bacterial capsular antigens, the methods and molecules of the present invention also apply to these other polysaccharide antigens. We believe it can be applied.
Accordingly, in one aspect, the invention relates to the identification of nucleic acid molecules useful for the detection and identification of specific bacterial polysaccharide antigens.
The present invention provides nucleic acid molecules derived from the following genes: genes encoding transferases; or genes encoding enzymes involved in the transport or processing steps of polysaccharide or oligosaccharide units, such as the wzx gene, wzy gene, or A gene having a similar function; said gene is involved in the synthesis of a particular bacterial polysaccharide antigen, in which case the sequence of the nucleic acid molecule is specific to the particular bacterial polysaccharide antigen.
Polysaccharide antigens such asE.coliCapsular antigens (type I and type II), toxic capsular of Salmoella sv typhi and species of capsular antigens such as Streptococcus pneumoniae and Staphylococcus albus , Encoded and provided by a gene including a sugar transferase gene and a gene for transport and processing steps of polysaccharide or oligosaccharide units. These are in some cases wzx or wzy, but in other cases they are quite different because different process steps are used. Examples of other gene clusters are Streptococcus thermophilus (Streptococcus thermophilus) Extracellular polysaccharide, Rhizobium merillotti (Rhizobiummelilotti)Exopolysaccharides, and Klebsiella pneumoniae (Klebsiella pneumoniae)This is a gene cluster for the K2 capsule and the like. All of these have genes that can be seen to include nucleotide sugar pathway genes, sugar transferase genes and genes for oligosaccharide or polysaccharide processing steps by experimental analysis, comparison of nucleotide sequences or predicted protein structures.
E.coli In the case of the K-12 colanic acid capsular gene cluster [Stevenson et al. (1996) "E.coli K-12 gene involved in the production of extracellular polysaccharide colanic acid" J. Bacteriol. 178 : 4885-4893] genes from three classes were tentatively or definitively identified. Cholanic acid capsules are classified as E. coli type I capsules.
The present inventors generally have a transferase gene and an oligosaccharide processing step gene that encodes a nucleotide sugar synthesis pathway such that most sugars present in such capsules occur in different serotype capsules, I believe it is more specific to a particular capsule. Thus, it can now be predicted that nucleotide sugar synthesis pathway genes are more general than those of the capsular type.
As described below, we have a polysaccharide antigen gene cluster that shares a transferase gene and / or oligosaccharide or polysaccharide gene, and therefore selects nucleotide sequences completely within these genes. That this may lead to cross-reactivity; an example of a capsular antigen isE.coli For type II capsules, only the transferase gene is sufficiently specific. However, we nevertheless, in light of their previous results, the present inventors have found that transferase genes or genes that control oligosaccharide or polysaccharide processing steps are responsible for the specific detection and characterization of polysaccharide serotypes. Therefore, we consider it a good target for selecting nucleotide sequences. Thus, if there is similarity between specific genes, selecting nucleotide sequences from other transferase genes or oligosaccharide or polysaccharide processing genes within the relevant gene cluster will still provide specificity. Wax, or using a combination of nucleotide sequences, will give the desired specificity. Combinations of nucleotide sequences may include both nucleotide sequences derived from pathway genes and nucleotide sequences derived from transferase, wzx or wzy genes.
Thus, the present invention also provides a group of nucleic acid molecules, which nucleic acid molecules are derived from a combination of genes encoding a transferase and / or polysaccharide or oligosaccharide unit transport or processing step enzyme, such as wzx or wzy. In this case, the combination of genes is specific for the synthesis of a particular bacterial polysaccharide antigen, in which case a group of nucleic acid molecules is specific for the bacterial polysaccharide antigen. In another preferred form, the nucleic acid molecule is derived together with a nucleic acid molecule derived from a pathway gene from a combination of genes encoding transferase and / or polysaccharide or oligosaccharide unit transport or processing step enzymes such as wzx or wzy. Is done.
In a second aspect, the present invention relates to the detection of bacteria expressing O antigens in diagnostic assays and the identification of nucleic acid molecules useful for identifying the O antigens of these bacteria.
The present invention provides nucleic acid molecules derived from the following genes: a gene encoding a transferase; or a gene encoding an enzyme involved in the transport or processing steps of a polysaccharide or oligosaccharide unit, such as a wzx or wzy gene; A gene is involved in the synthesis of a specific bacterial O antigen, in which case the sequence of the nucleic acid molecule is specific for a particular bacterial O antigen.
The length of the nucleic acid of the present invention may vary. In one embodiment, the length of the nucleic acid consists of about 10 to about 20 nucleotides.
In one preferred embodiment, the present invention provides nucleic acid molecules derived from the following genes: genes encoding transferases; or genes encoding enzymes involved in transport or processing steps of polysaccharide or oligosaccharide units, such as A wzx or wzy gene, which is involved in the synthesis of O antigen expressed by E. coli, and its nucleic acid molecule sequence is specific to O antigen.
In a further preferred embodiment, the sequence of the nucleic acid molecule is specific for the nucleotide sequence encoding the O111 antigen (SEQ ID NO: 1). More preferably, the sequence is wbdH (nucleotide positions 739-1932 of SEQ ID NO: 1), wzx (nucleotide positions 8646-9911 of SEQ ID NO: 1), wzy (nucleotide positions 9901 to 10953 of SEQ ID NO: 1), wbdM (SEQ ID NO: 1). 1 nucleotide position 11821-12945), and a gene selected from the group consisting of fragments of these molecules at least 10-12 nucleotides in length. Particularly preferred nucleic acid molecules are those listed in Tables 5 and 5A for the above genes.
In another further preferred embodiment, the sequence of the nucleic acid molecule is specific for the nucleotide sequence encoding the O157 antigen (SEQ ID NO: 2). More preferably, the sequence is wbdN (nucleotide positions 79 to 861 of SEQ ID NO: 2), wbdO (nucleotide positions 2011 to 2757 of SEQ ID NO: 2), wbdP (nucleotide positions 5257 to 6471 of SEQ ID NO: 2), wbdR (SEQ ID NO: 2). 2 nucleotide positions 13156 to 13821), wzx (nucleotide positions 2744 to 4135 of SEQ ID NO: 2), and wzy (nucleotide positions 858 to 2042 of SEQ ID NO: 2). Particularly preferred nucleic acid molecules are those listed in Tables 6 and 6A.
In a further preferred embodiment, the present invention provides a nucleic acid molecule derived from the following gene: a gene encoding a transferase; or a gene encoding an enzyme involved in transport or processing steps of a polysaccharide or oligosaccharide unit, for example, wzx or wzy gene;S.entericaIn which the sequence of the nucleic acid molecule is specific for the O antigen.
In a more preferred form of this embodiment, the sequence of the nucleic acid molecule isS.enterica Specific for nucleotide sequence encoding C2 antigen (SEQ ID NO: 3). More preferably, the sequence of the nucleic acid molecule is derived from a gene selected from the group consisting of the following genes: wbaR (nucleotide positions 2352 to 3314 of SEQ ID NO: 3), wbaL (nucleotide positions 3361 to 3875 of SEQ ID NO: 3). ), WbaQ (nucleotide positions 3777-5020 of SEQ ID NO: 3), wbaW (nucleotide positions 6313-7323 of SEQ ID NO: 3), wbaZ (nucleotide positions 7310-8467 of SEQ ID NO: 3), wzx (nucleotide positions 1019 of SEQ ID NO: 3). ˜2359), and wzy (nucleotide positions 5114-6313 of SEQ ID NO: 3). Particularly preferred nucleic acid molecules are those listed in Table 7.
In a more preferred further form of this embodiment, the sequence of the nucleic acid molecule isS.entericaSpecific for nucleotide sequence encoding B antigen (SEQ ID NO: 4). More preferably, the sequence is derived from wzx (nucleotide positions 12762 to 14054 of SEQ ID NO: 4) or wbaV (nucleotide positions 14059 to 15060 of SEQ ID NO: 4). Particularly preferred nucleic acid molecules are those listed in Table 8 derived from the wzx and wbaV genes.
In an even more preferred form of this embodiment, the sequence of the nucleic acid molecule is specific for the S. enterica D3O antigen and is derived from the wzy gene.
In an even more preferred form of this embodiment, the sequence of the nucleic acid molecule isS.entericaIt is specific for the E1 O antigen and is derived from the wzx gene.
Transfer genes or genes encoding transport or processing steps of polysaccharide or oligosaccharide units, such as wzx or wzy genes, are excellent targets for specific detection of individual O serotypes, but differ such that cross-reactions occur Within this group are individual genes or portions thereof that can prove to be identical or closely related between O serotypes. Cross-reactions should be avoided by selecting different targets within this group or by using multiple targets within this group.
Furthermore, the O antigen gene cluster may arise from the recombination of at least two strains such that a unique O antigen type is provided by a combination of gene products shared with at least two other O antigen types. Is recognized. Examples of this phenomenon areS.entericaO antigen serotype D2, which has genes from D1 and E1, but none of D2 is unique. In these situations, detection of the O antigen type can still be achieved according to the present invention, but in order to detect a unique combination of genes that are present only in this particular O antigen gene cluster, a combination of nucleic acid molecules is used. There is a need to.
Thus, the present invention also provides a group of nucleic acid molecules, wherein the nucleic acid molecule is a gene encoding an enzyme for transport or processing steps of a transferase and / or polysaccharide or oligosaccharide unit, such as wzx or Derived from the wzy gene, in which case a group of nucleic acid molecules are specific for the bacterial O antigen. Preferably, the specific bacterial O antigen isS.entericaExpressed by More preferably, the group of nucleic acid molecules is specific for the D2O antigen and is derived from the E1 wzy gene and the D1 wzx gene.
A combination of nucleotide sequences may include a nucleotide sequence derived from a pathway gene, together with a nucleotide sequence derived from a transferase, a wzx or wzy gene.
Thus, the present invention also provides a group of nucleic acid molecules, wherein the nucleic acid molecule is a gene encoding an enzyme for transport or processing steps of a transferase and / or polysaccharide or oligosaccharide unit, such as wzx or Derived from the wzy gene, and the sugar pathway gene, in which case a group of nucleic acid molecules are specific for the bacterial O antigen. Preferably, the O antigen isS.entericaExpressed by
It is further observed that pseudohybridization may occur through the initial selection of sequences found in many different genes, which typically compares, for example, the size of the band to a PCR gel control. In that case, other alternative sequences can be selected.
We are based on the knowledge of the present invention and information available on polysaccharide antigen gene clusters (including O antigen gene clusters) and through the use of experimental analysis, comparison of nucleic acid sequences or predicted protein structures. The nucleic acid molecule according to the present invention can be easily induced toward a specific polysaccharide antigen of interest. Appropriate bacterial strains are generally available for a fee from the depository.
As mentioned above, there are currently 166 types confirmedE.coliWhile there is O antigen,S.entericaHas 46 known O antigen types [Popoff M. Y. et al. (1992) "Salmonella subtype antigen form" 6th edition,S.entericaWHO Collaborating Center for Reference and Research, Pasteur Institute, Paris France]. Many other bacterial genera are known to have the O antigen, which are Citrobacter, Shigella, Yersinia, Plesiomonas, Vibrio and Proteus. ).
166 different typesE.coli  Samples of the O antigen serotype are available from the National Serum Institute, Copenhagen, Denmark.
46 kindsS.entericaSerotypes are available from the Institute of Medicine and Veterinary Medicine, Adelaide, Australia.
In another aspect, the invention relates to a method of testing a sample for the presence of one or more bacterial polysaccharide antigens, the method contacting the sample with at least one oligonucleotide molecule that can specifically hybridize to the following genes: Consisting of: (i) a gene encoding a transferase, or (ii) a gene encoding an enzyme for transport or processing steps of an oligosaccharide or polysaccharide unit, such as a wzx or wzy gene; Relevant to the synthesis of polysaccharide antigens; suitable conditions are that at least one oligonucleotide molecule specifically hybridizes to at least one such gene of a bacterium expressing a particular bacterial polysaccharide antigen present in the sample; A specifically hybridized oligonucleotide molecule It is a condition to be able to leave.
If a single specific oligonucleotide molecule is not available, a combination of molecules that specifically hybridize to the target region may be used. Thus, the present invention provides a group of nucleic acid molecules for use in the test methods of the present invention, wherein the nucleic acid molecules are genes encoding transferase and / or polysaccharide or oligosaccharide unit transport or processing step enzymes. Derived from, for example, wzx or wzy, where a group of nucleic acid molecules are specific for a particular bacterial polysaccharide. A group of nucleic acid molecules can optionally include nucleic acid molecules derived from sugar pathway genes.
In another aspect, the invention relates to a method for testing a sample for the presence of one or more bacterial polysaccharide antigens, the method specifically hybridizing the sample having at least one pair of oligonucleotide molecules to the following genes: Contacting with at least one oligonucleotide molecule of said pair of: (i) a gene encoding a transferase, or (ii) a gene encoding an enzyme for transport or processing steps of an oligosaccharide or polysaccharide unit, For example, a wzx or wzy gene; where the gene is involved in the synthesis of a bacterial polysaccharide antigen; appropriate conditions are that at least one oligonucleotide molecule of the counter molecule expresses a particular bacterial polysaccharide antigen present in the sample Specific to at least one such gene in the bacterium To hybridize, a condition that specifically allows detection of the oligonucleotide molecule hybridized.
Paired oligonucleotide molecules may both hybridize to the same gene or to different genes. Only one oligonucleotide molecule in the pair needs to specifically hybridize to a sequence specific for a particular antigen type. Other molecules can hybridize to non-specific regions.
If a specific polysaccharide antigen gene cluster is generated through recombination, at least one pair of oligonucleotide molecules can hybridize to a specific combination of genes in the cluster specific for that polysaccharide antigen. Alternatively, multiple pairs may be selected to hybridize to a particular combination of genes. Even if the genes in a particular cluster are all unique, the method can be carried out using nucleotide molecules that recognize the combination of genes within the cluster.
Thus, the present invention provides a group comprising nucleic acid molecule pairs for use in the test methods of the present invention, wherein the nucleic acid molecule pairs are transported or processed by transferase and / or polysaccharide or oligosaccharide units. Derived from genes encoding enzymes for, for example, wzx or wzy genes, wherein a group of nucleic acid molecules are specific for a particular bacterial polysaccharide antigen. A group of nucleic acid molecules can optionally include pairs of nucleic acid molecules derived from sugar pathway genes.
In another aspect, the invention relates to a method for testing a sample for the presence of one or more specific bacterial O antigens, the method comprising at least one oligo capable of specifically hybridizing the sample to the following genes: Consisting of contacting with a nucleotide molecule: (i) a gene encoding an O antigen transferase, or (ii) a gene encoding an enzyme for transport or processing steps of an oligosaccharide or polysaccharide unit, such as a wzx or wzy gene; In this case, the gene is involved in the synthesis of a specific O antigen; appropriate conditions are that at least one oligonucleotide molecule is present in at least one such gene of a bacterium that expresses the specific bacterial O antigen present in the sample. Specifically hybridized and specifically hybridized oligonucleotides It is a condition to be able to detect a fault molecule. Preferably, the bacteriaE.coliOrS.entericaIt is. More preferably,E.coliExpresses the O157 or O111 serotype. More preferably,S.entericaExpresses C2 or B serotype. Preferably, this method is a Southern blot method. More preferably, the nucleic acid molecule is labeled and the hybridization of the nucleic acid molecule is detected by autoradiography or fluorescence detection.
We envisage the case where a single specific oligonucleotide molecule is not available. In such cases, a combination of molecules that specifically hybridize to the target region may be used. Thus, the present invention provides a group of nucleic acid molecules for use in the test methods of the present invention, wherein the nucleic acid molecules comprise enzymes for transport and / or transport or processing steps of polysaccharide or oligosaccharide units. Derived from the encoding gene, eg, the wzx or wzy gene, wherein a group of nucleic acid molecules are specific for a particular bacterial O antigen. Preferably, the specific bacterial O antigen isS.entericaExpressed by A group of nucleic acid molecules can optionally include nucleic acid molecules derived from sugar pathway genes.
In another aspect, the invention relates to a method of testing a sample for the presence of one or more specific bacterial O antigens, the method specifically hybridizing the sample having at least one pair of oligonucleotide molecules to the following genes: Contact with at least one oligonucleotide molecule of the pair capable of: (i) encoding a gene encoding an O antigen transferase, or (ii) encoding an enzyme for transport or processing steps of an oligosaccharide or polysaccharide unit A gene that performs, for example, a wzx or wzy gene; where the gene is involved in the synthesis of a specific O antigen; appropriate conditions are that at least one oligonucleotide molecule expresses a specific bacterial O antigen present in the sample Specifically for at least one such gene in bacteria And hybrid formation is a condition that specifically allows detection of the oligonucleotide molecule hybridized.
Preferably, the bacteriaE.coliOrS.entericaIt is. More preferably,E.coliAre of the O111 or O157 serotype. More preferably,S.entericaExpresses C2 or B serotype. Preferably, the method is a polymerase chain reaction method. More preferably, the oligonucleotide molecule used in the method of the present invention is labeled. Even more preferably, the hybridized oligonucleotide molecule is detected by electrophoresis. Preferred oligonucleotides used for O111 are those for specific detection of O111, which are listed in Tables 5 and 5A in relation to genes wbdH, wzx, wzy and wbdM. Preferred oligonucleotides used for O157 are those for O157 specific detection methods, which are listed in Tables 6 and 6A.
For serotypes C2 and B, suitable oligonucleotide molecules can be selected from the appropriate regions listed in column 3 of Tables 7 and 8.
We rarely, but two genetically similar gene clusters encoding serologically different O antigens, are generated through genetic recombination or mutation to produce polymorphic variants. Assume that there are cases. In such a case, multiple pairs of oligonucleotides are selected and hybridized to specifically combined genes. Thus, the present invention provides a group comprising nucleic acid molecule pairs for use in the test methods of the present invention, wherein the nucleic acid molecule pairs are transported or processed by transferase and / or polysaccharide or oligosaccharide units. Derived from genes encoding enzymes for, for example, wzx or wzy genes, wherein a group of nucleic acid molecules is specific for a particular bacterial O antigen. Preferably, the specific bacterial O antigen isS.entericaExpressed by A group of nucleic acid molecules can optionally include pairs of nucleic acid molecules derived from sugar pathway genes.
In another aspect, the present invention relates to a method for testing a food-derived sample for the presence of one or more specific bacterial O antigens, wherein the method specifically targets the sample having at least one pair of oligonucleotide molecules to the following genes: For contacting with at least one oligonucleotide molecule of the pair capable of hybridizing: (i) a gene encoding an O antigen transferase, or (ii) transport or processing steps of an oligosaccharide or polysaccharide unit A gene encoding an enzyme, such as a wzx or wzy gene; where the gene is involved in the synthesis of a particular O antigen; suitable conditions are a specific bacterial polysaccharide in which at least one oligonucleotide molecule is present in the sample Specific to at least one such gene of the bacterium expressing the antigen To hybridize, a condition that specifically allows detection of the oligonucleotide molecule hybridized. Preferably, the bacteriaE.coliOrS.entericaIt is. More preferably,E.coliAre of the O111 or O157 serotype. More preferably,S.entericaAre of the C2 or B serotype. Preferably, the method is a polymerase chain reaction method. More preferably, the oligonucleotide molecule used in the method of the present invention is labeled. Even more preferably, the hybridized oligonucleotide molecule is detected by electrophoresis.
In another aspect, the invention relates to a method of testing a stool-derived sample for the presence of one or more specific bacterial O antigens, the method specifically hybridizing the sample having at least one pair of oligonucleotide molecules to the following genes: Contacting with at least one oligonucleotide molecule of the pair that can be formed: (i) a gene encoding an O antigen transferase, or (ii) an enzyme for transport or processing steps of an oligosaccharide or polysaccharide unit A gene that encodes, for example, a wzx or wzy gene; in this case, the gene is involved in the synthesis of a specific O antigen; appropriate conditions are that at least one oligonucleotide molecule is a specific bacterial O antigen present in a sample. Specific to at least one of the genes of the bacteria to be expressed And Brides formed, it is a condition that specifically allows detection of the oligonucleotide molecule hybridized. Preferably, the bacteriaE.coliOrS.entericaIt is. More preferably,E.coliAre of the O111 or O157 serotype. More preferably,S.entericaAre of the C2 or B serotype. Preferably, the method is a polymerase chain reaction method. More preferably, the oligonucleotide molecule used in the method of the present invention is labeled. Even more preferably, the hybridized oligonucleotide molecule is detected by electrophoresis.
In another aspect, the invention relates to a method for testing a patient-derived sample for the presence of one or more specific bacterial O antigens, the method specifically hybridizing the sample having at least one pair of oligonucleotide molecules to the following genes: Contacting with at least one oligonucleotide molecule of the pair that can be formed: (i) a gene encoding an O antigen transferase, or (ii) an enzyme for transport or processing steps of an oligosaccharide or polysaccharide unit A gene that encodes, for example, a wzx or wzy gene; in this case, the gene is involved in the synthesis of a specific O antigen; appropriate conditions are that at least one oligonucleotide molecule is a specific bacterial O antigen present in a sample. Specific to at least one such gene of the expressing bacteria And hybrid formation is a condition that specifically allows detection of the oligonucleotide molecule hybridized. Preferably, the bacteriaE.coliOrS.entericaIt is. More preferably,E.coliAre of the O111 or O157 serotype. More preferably,S.entericaAre of the C2 or B serotype. Preferably, the method is a polymerase chain reaction method. More preferably, the oligonucleotide molecule used in the method of the present invention is labeled. Even more preferably, the hybridized oligonucleotide molecule is detected by electrophoresis.
It is understood in the above method that when using a pair of oligonucleotides, one of the oligonucleotide sequences hybridizes to a sequence that is not from a transferase, wzx or wzy gene. Furthermore, if both hybridize to one of these gene products, they can hybridize to the same or different of these genes.
It is further understood that if cross-reactivity is a problem, oligonucleotide combinations may be selected and gene combinations detected to provide specificity.
Furthermore, the present invention relates to a diagnostic kit which can be used for detection of bacteria expressing bacterial polysaccharide antigens and identification of bacterial polysaccharide types of these bacteria.
Thus, in a further aspect, the invention relates to a kit comprising a first vial containing a first nucleic acid molecule capable of specifically hybridizing to the following gene: (i) a gene encoding a transferase, or ( ii) Genes encoding enzymes for oligosaccharide or polysaccharide transport or processing steps, eg wzx or wzy genes; in this case, the genes are involved in the synthesis of bacterial polysaccharides. The kit also provides a second specific nucleic acid that can specifically hybridize to the following genes in the same or separate vials: (i) a gene encoding a transferase, or (ii) an oligosaccharide or polysaccharide A gene encoding an enzyme for transport or processing steps, such as the wzx or wzy gene; in this case, the gene is involved in the synthesis of bacterial polysaccharide, wherein the sequence of the second nucleic acid molecule is the sequence of the first nucleic acid molecule Is different.
In a further aspect, the present invention relates to a kit comprising a first vial containing a first nucleic acid molecule capable of specifically hybridizing to the following gene: (i) a gene encoding a transferase, or (ii) an oligosaccharide Or a gene encoding an enzyme for the transport or processing step of a polysaccharide, such as the wzx or wzy gene; in this case, the gene is involved in the synthesis of the bacterial O antigen. The kit also provides a second specific nucleic acid that can specifically hybridize to the following genes in the same or separate vials: (i) a gene encoding a transferase, or (ii) an oligosaccharide or polysaccharide A gene encoding an enzyme for transport or processing steps, such as the wzx or wzy gene; in this case, the gene is involved in the synthesis of the O antigen, in which case the sequence of the second nucleic acid molecule is the sequence of the first nucleic acid molecule Are different. Preferably the first and second nucleic acid sequences areE.coliOr the first and second nucleic acid sequences are derived fromS.entericaIt was derived from.
The present inventors have provided the full-length sequence of the O157 gene cluster for the first time, generated a recombinant molecule from this sequence of the entire gene cluster that has not been cloned so far, inserted for expression, and applied to vaccines, etc. Recognizing that it is possible to provide useful expression O157.
Definition
The phrase “nucleic acid molecule derived from a gene” means that the nucleic acid molecule has a nucleotide sequence that is identical or substantially similar to all or part of the identified gene. Thus, a nucleic acid molecule derived from a gene is identical or substantially identical to a molecule isolated by physical separation from the identified gene, or all or part of an artificially synthesized and identified gene. It may be a molecule having a substantially similar nucleotide sequence. Some researchers consider only DNA strands that have the same sequence as the mRNA transcribed from the gene, but here both strands are intended.
A transferase gene is a region of a nucleotide sequence-bearing nucleic acid that encodes a gene product for monomeric sugar unit transport.
The flippase or wzx gene is a region of a nucleotide sequence-bearing nucleic acid that encodes a gene product that moves an oligosaccharide repeat unit, typically composed of 3 to 6 monomeric sugar units, to the outer surface of the membrane.
The polymerase or wzy gene is a region of a nucleotide sequence-carrying nucleic acid that encodes a gene product that polymerizes repeating oligosaccharide units, typically consisting of 3 to 6 monomeric sugar units.
The nucleotide sequences provided herein are set forth in the sequences listed as antisense sequences. This term is in the same manner as used in the Biochemical Glossary and Molecular Biology, revised edition, David M. Glick, 1997, Portland Press, London, page 11. Used; in the literature, this term is described as referring to one of the duplexes of double-stranded DNA, but usually it has the same sequence as mRNA. We use this to describe this strand with the same sequence as the mRNA.
Nomenclature
E.coli Synonyms of O111rfb
Current name            Inventor naming          Bastin et al. 1991
wbdH orf 1
gmd orf 2
wbdI orf 3 orf 3.4 *
manC orf 4 rfbM *
manB orf 5 rfbK *
wbdJ orf 6 orf 6.7 *
wbdK orf 7 orf 7.7 *
wzx orf 8 orf 8.9 and rfbX *
wzy orf 9
wbdL orf 10
wbdM orf 11
^*The nomenclature is Bastin D. A. et al., 1991, “E.coli O antigen determination rfb gene cluster of O111 strainE.coli Molecular cloning and expression at K-12 ", Mol. Microbiol. 5: 9, 22223-2231.
Other synonyms
wzy rfc
wzx rfbX
rmlA rfbA
rmlB rfbB
rmlC rfbC
rmlD rfbD
glf orf 6 *
wbbI orf 3 #,E.coli K-12 orf 8 *
wbbJ orf 2 #,E.coli K-12 orf 9 *
wbbK orf l #,E.coli K-12 orf 10 *
wbbL orf 5 #,E.coli K-12 orf 11 *
# Named according to Yao, Z. and M. A. Valvano 1994.
"E.coli Genetic analysis of K-12 W3110 O-specific lipopolysaccharide biosynthetic region (rfb): identification of genes conferring group specificity to Shigella flexneri serotypes Y and 4a ". J. Bacteriol. 176: 4133-4143.
* Naming is according to Stevenson et al., 1994. "E.coli Structure of K-12 O antigen and its rfb gene cluster sequence ". J. Bacteriol. 176: 4144-4156.
・S.entericaIs the name adopted in 1987 and has been replaced by many other names, such as Salmonella typhi and Salmonella typhimurium. The traditional species name isS.enterica Serovar names such as sv Tiffy. But traditional names are still widely used.
• Many species of O antigen genes have been given rfb names (such as rfbA), and O antigen gene clusters have often been referred to as rfb clusters. The new names for the rfb gene are as shown in the table. In this specification, both terms are used according to the information source.
[Brief description of the drawings]
Figure 1E.coli Cosmid clones pPR1054, pPR1055, pPR1056, pPR1058, pPR1287, which are subclones of the O111 O antigen gene clusterE.coli An R1 restriction map is shown. The thick line is a region common to all clones. A broken line shows the discontinuous part on a chromosome.E.coliAn estimated restriction map of strain M92 is shown above.
Figure 2 shows cosmid clone pPR1058.E.coli Figure 3 shows a restriction mapping analysis of the O111 O antigen gene cluster. Restriction enzymes are as follows: B: Bam1; Bg: Bg1II; E: E.coR1; H: HindIII; K: KpnI; P: PstI; S: Sa1I; and X: Xho1. Plasmids pPR1230, pPR1231, and pPR1288 are deleted derivatives of pPR1058. Plasmids pPR1237, pPR1238, pPR1239 and pPR1240 are in pUC19. Plasmids pPR1243, pPR1244, pPR1245, pPR1246 and pPR1248 are in pUC18 and pPR1292 is in pUC19. Plasmid pPR1270 is in pT7T319U. Probes 1, 2 and 3 were isolated as internal fragments of pPR1246, pPR1243 and pPR1237, respectively. The dotted line indicates that the subclone DNA extends to the left of the map and is bound to the vector.
Figure 3E.coli The structure of the O111 O antigen gene cluster is shown.
Figure 4E.coli The structure of the O157 O antigen gene cluster is shown.
Figure 5 encodes the serogroup C20 O antigen gene clusterS.entericaThe structure of the locus is shown.
Figure 6 encodes the serogroup BO antigen gene clusterS.entericaThe structure of the locus is shown.
FIG.E.coli The nucleotide sequence of the O111 O antigen gene cluster is shown. Notes: (1) The leading and trailing three bases of the gene are underlined and italicized respectively; (2) Bastin and Reeves (1995), “E.coliO111 O antigen gene (rfb) cluster sequence and analysis ", Gene 164: 17-23), marks the region previously sequenced.
Figure 8E.coli The nucleotide sequence of the O157 O antigen gene cluster is shown. Notes: (1) The leading and trailing three bases of the gene are underlined and italicized respectively; (2) Bilge et al. [1996, “In Adhesion”E.coliThe role of the O157-H7 side chain and analysis of the rfb locus "Inf. And Immun. 64: 4795-4801] are marked in regions previously sequenced.
Figure 9S.entericaThe nucleotide sequence of a serogroup C2O antigen gene cluster is shown. Note: (1) The assigned number is according to Brown et al. (1992). "Molecular analysis of the rfb gene cluster of Salmonella subtype muenchen (strain M67): genetic basis of polymorphism between groups C2 and B", Mol. Microbiol. 6: 1385-1394. (2) The three bases at the beginning and end of the gene are shown in underlined and italicized respectively. (3) Only the portion of the group C2 gene cluster that differs from group B is sequenced and presented here.
FIG.S.entericaThe nucleotide sequence of a serogroup BO antigen gene cluster is shown. (1) The assignment number is according to Jiang et al. (1991). "S.entericaStructure and sequence of rfb (O antigen) gene cluster of blood type subtype typhimurium (strain LT2) ", Mol. Microbiol. 5: 695-713. The first gene of the O antigen gene cluster is rm1B starting at base 4099. (2) The three bases at the beginning and end of the gene are shown in underlined and italicized respectively.
Best method for carrying out the present invention
Materials and Methods-Part 1
E.coli Experimental procedures for isolation and characterization of the O111O antigen gene cluster (positions 3,021-9,981) are described in Bastin D.A. et al., 1991 “E.coli Molecular cloning of the rfb gene cluster that determines the O antigen of the O111 strain andEscherichia coli Expression in K-12 "Mol. Microbiol. 5: 9 2223-2231, and Bastin D.A. and Reeves, P.R. 1995"Escherichia coli Sequence and analysis of the O111 O antigen gene (rfb) group "according to Gene 164: 17-23.
A. Bacterial strains and growth media
Bacteria were grown in Luria broth supplemented as needed.
B. Cosmids and phages
The cosmid in host strain x2819 was repackaged in vivo. Cells were grown to an optimal density of 0.3 at 580 nm in a 250 mL flask containing 30 mL culture at 30 ° C. with moderate shaking. The defective λ prophage was induced by heating in a water bath for 15 minutes at 45 ° C. followed by incubation for 2 hours at 37 ° C. with vigorous shaking. Cells were then lysed by adding 0.3 mL chloroform and shaking for an additional 10 minutes. Cell debris was removed from 1 mL of lysate by spinning for 5 minutes in a microcentrifuge and the supernatant was collected in a new microcentrifuge tube. A drop of chloroform was added and then the entire tube contents were stirred vigorously.
C. DNA preparation
Chromosomal DNA was prepared from bacteria grown overnight at 37 ° C. in a 30 mL volume of Luria broth. Cells were collected by centrifugation, washed and resuspended in 10 mL of 50 mM Tris-HCl pH 8.0. EDTA was added and the mixture was incubated for 20 minutes. Lysozyme was then added and incubation continued for an additional 10 minutes. Proteinase K, SDS, and ribonuclease were then added and the mixture was incubated for 2 hours when lysis occurred. All incubations were at 37 ° C. The mixture was then heated to 65 ° C. and extracted once with 8 mL of phenol at the same temperature. The mixture was extracted once with 4 mL of phenol / chloroform / isoamyl alcohol at 4 ° C. Residual phenol was removed by two ether extractions. DNA was precipitated with 2 volumes of ethanol at 4 ° C., spooled and washed in 70% ethanol, resuspended in 1-2 mL TE and dialyzed. Plasmid and cosmid DNA were modified by the Bironboim and Doly method [Birnboim, HC and Doly, J. (1979) Rapid alkaline extraction treatment Nucl. Acid Res. 7: 1513-1523] to screen recombinant plasmid DNA. Prepared. The culture volume was 10 mL and the lysate was extracted with phenol / chloroform / iso-amyl alcohol and then precipitated with isopropanol. Plasmid DNA used as a vector was isolated on a continuous cesium chloride gradient after alkaline lysis of cells grown in 1 L culture.
D. Enzymes and buffers
Restriction endonucleases and DNA T4 ligase were purchased from Boehringer Mannheim (Castle Hill, NSW, Australia) or Pharmacia LKB (Melboume, VIC Australia). Restriction enzymes were used in the recommended commercial buffer.
E. Gene bank construction
M92 chromosomal DNA (Stoke W strain, Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen S, Denmark) was partially digested with 0.2U Sau3A1 for 1-15 minutes. An aliquot of the largest but about 40-50 kb fragment in size was selected and ligated into the vector pPR691, previously digested with BamHI and PvuII. The ligation mixture was packaged in vitro with the packaging extract. The host strain for transduction was x2819 and the recombinant was selected with kanamycin.
F. Serological procedure
Colonies were screened for the presence of O111 antigen by immunoblotting. Colonies were grown overnight to 100 per plate, then transferred to nitrocellulose discs and lysed with 0.5N HCl. Tween 20 was added to TBS at a final concentration of 0.05% for the blocking, incubation, and washing steps. Primary antibody diluted 1: 800E.coli Group O was 111 antiserum. The secondary antibody was goat anti-rabbit IgG labeled with horseradish peroxidase diluted 1: 5000. The staining substrate was 4-chloro-1-naphthol. Slide aggregation was performed according to standard procedures.
G. Recombinant DNA method
Restriction mapping was based on a combination of standard methods including single and double stranded digestion and subcloning. A complete cosmid deletion derivative was generated as follows: An aliquot of 1.8 μg cosmid DNA was digested with 0.25 U restriction enzyme in a volume of 20 μl for 5-80 minutes. One half of each aliquot was used to check the extent of digestion on an agarose gel. Samples that seem to give a representative range of fragments were ligated overnight at 4 ° C and CaCl₂JM109 was transformed by the method. The selected plasmid was transformed into sφ174 by the same method. P4657 was transformed with pPR1244 by electroporation.
H. DNA hybridization
Probe DNA was extracted from an agarose gel by electroelution and nick translated using [α-32P] -dCTP. Chromosomal DNA or plasmid DNA was electrophoresed on a 0.8% agarose gel and transferred to a nitrocellulose membrane. Hybridization buffer and prehybridization buffer contained 30% or 50% formamide for low or high stringency probing, respectively. Incubation temperatures were 42 ° C and 37 ° C for prehybridization and hybridization, respectively. The low stringency wash of the filter consisted of a 3 x 20 minute wash in 2 x SSC and 0.1% SDS. High stringency washes include 3 x 5 min washes in 2 x SSC and 0.1% SDS at room temperature, 1 h washes in 1 x SSC and 0.1% SDS at 58 ° C, and 0.1 at 58 ° C Consists of 15 minutes wash in xSSC and 0.1% SDS.
I.E.coli Nucleotide sequencing of the O111O antigen gene cluster (positions 3,021-9,981)
Nucleotide sequencing was performed using an ABI 373 automated sequencer (CA, USA). The region between the 3.30 and 7.90 map positions was sequenced using a deletion family unidirectional exonuclease III digestion made in PT7T3190 from clones pPR1270 and pPR1272. The gap was mainly filled by cloning the selected fragment into M13mp18 or M13mp19. The region from the location of the map from 7.10 to 10.2 was sequenced from restriction fragments in M13mp18 or M13mp19. The remaining gap in both regions was filled by starting with a synthetic oligonucleotide complementary to the position determined along the sequence using a single stranded DNA template in M13 or phagemid. Oligonucleotides were designed after analyzing adjacent sequences. All sequencing was performed by the chain termination method. Sequences are described in SAP [Staden, R., 1982 “Automated computer manipulation of gel reading data generated by the shotgun method of DNA sequencing” Nuc. Acid Res. 10: 4731-4751; Staden, R., 1986 “ Aligned using the current state and portability of our sequence manipulation software “Nuc. Acid Res. 14: 217-231”. Using the program NIP [Staden, R. 1982 “Interactive Graph Program for Comparing and Aligning Nucleic Acids and Amino Acid Sequences” Nuc. Acid Res. 10: 2951-2961], finding open reading frames, and These were translated into proteins.
J. et al.E.coli Isolation of clones carrying the O111 O antigen gene cluster
E.coli The O antigen gene cluster was analyzed by the method of Bastin D.A. et al. [1991 “E.coli Molecular cloning of rfb gene cluster determining O antigen of O111 strain and expression in Escherichia coli K-12 "Mol. Microbiol. 5 (9) 2223-2231]. A cosmid gene bank of M92 chromosomal DNA was established in in vivo packaging strain x2819. From gene bank, 3.3 × 10^ThreeColonies using an immunoblotting procedureE.coli Screened with O111 antiserum: 5 colonies (pPR1054, pPR1055, pPR1056, pPR1058, and pPR1287) were positive. Cosmids from these strains are packaged in vivo into lambda particles and lack all O antigen genesE.coliThe deletion mutant Sφ174 was transduced. In this host strain, all plasmids gave positive aggregation with O111 antiserum. The Eco R1 restriction map of 5 independent cosmids showed that they commonly have a region of about 11.5 kb (FIG. 1). Cosmid pPR1058 was selected for analysis of the O antigen gene cluster region because it contained sufficient flanking DNA to identify several chromosomal markers linked to the O antigen gene cluster.
K. Restriction mapping of cosmid pPR1058
Cosmid pPR1058 was mapped in two stages. A preliminary map was first created and then the region between the map positions from 0.00 to 23.10 was mapped in detail. Because this has been shown to be sufficient for O111 antigen expression. The restriction sites for both stages are shown in FIG. The region common to the five cosmid clones was between the map positions of pPR1058 from 1.35 to 12.95.
To locate the O antigen gene cluster within pPR1058, pPR1058 cosmid isS.enterica LT2 andE.coli Probed with a DNA probe covering the region adjacent to the O antigen gene cluster from K-12. The capsular polysaccharide (cps) gene is upstream of the O antigen gene cluster, while the gluconate dehydrogenase (gnd) gene and the histidine (his) operon are downstream, the latter being further away from the O antigen gene cluster. . The probes used were pPR472 (3.35 kb) carrying the LT2 gnd gene, pPR685 (5.3 kb) carrying the cpsB and cpsG genes of the LT2 cps group, and all of the K-12 his operon. It was K350 (16.5 kb). The probe hybridized as follows: pPR472 is located at a map position from 12.95 to 15.1, 1.55 kb of PstI and HindIII double digestion of pPR1246 (HindIII / EcoRI subclone derived from pPR1058, FIG. 2) And hybridize to the 3.5 kb (including 2.7 kb vector) fragment; pPR685 hybridizes to the 4.4 kb EcoRI fragment (including the 1.3 kb vector) of pPR1058 located at the map position from 0.00 to 3.05; K350 Hybridized with a 32 kb EcoRI fragment of pPR1058 (including a 4.0 kb vector) located at the map position of 17.3 to 45.90. The subclone containing the putative gnd region is gnd^-edd^-Supplemented stock GB23152. In bromothymol gluconate blue plates, pPR1244 and pPR1292 in this host strain⁺edd^-A green colony with a predicted genotype was given. his⁺The phenotype was restored by plasmid pPR1058 in his-deficient strain Sφ174 in minimal media plates, indicating that the plasmid carries the complete his operon.
The O antigen gene cluster region isE.coliandS.entericaAs in the strain, it appears to be between gnd and cps, and therefore between approximately 3.05 and 12.95 map positions. To confirm this, a deletion derivative of pPR1058 was made as follows: First, pPR1058 was partially digested with HindIII and self-ligated. Transformants were selected for kanamycin resistance and screened for expression of the O111 antigen. Two colonies gave a positive response. EcoRI digestion showed that the two colonies assembled the same plasmid, one of which was called pPR1230, with an insert extending to the map position of 0.00 to 23.10. Second, pPR1058 was digested with SalI and partially digested with XhoI, and the compatible ends were religated. Transformants were selected with kanamycin and screened for O111 antigen expression. The plasmid DNA of eight positively reacting clones was confirmed using EcoRI and XhoI digestion and appeared identical. One cosmid was designated pPR1231. The insert of pPR1231 contained a DNA region between 0.00 to 15.10 map positions. Third, pPR1231 was partially digested with XhoI, self-ligated, and transformants were selected on streptomycin / streptomycin plates. Clones were screened for kanamycin sensitivity and 10 were selected, all of which deleted the DNA region from the XhoI site in the vector to the XhoI site at position 4.00. These clones do not express the O111 antigen and the XhoI site at position 4.00 is within the O antigen gene cluster. One clone was selected and named pPR1288. Plasmids pPR1230, pPR1231, and pPR1288 are shown in FIG.
L.E.coli Analysis of O111O antigen gene cluster (positions 3,021-9,981) nucleotide sequence data
Bastin and Reeves [1995Escherichia coli Sequence and analysis of the O111 O antigen gene (rfb) group "Gene 164: 17-23], in part, by sequencing fragments from the map positions 3,021-9,981E.coli The O111O antigen gene cluster was characterized. FIG.E.coli The gene organization at positions 3,021 to 9,981 of the O111O antigen gene cluster is shown. orf3 and orf6 have a very high level of amino acid identity with wcaH and wcaG (46.3% and 37.2%, respectively) and in functionE.coli It appears to be similar to the sugar biosynthetic pathway genes in the K-12 colanic gene cluster. orf4 and orf5 show high levels of amino acid homology to the manC and manB genes, respectively. orf7 shows a high level of homology with rfbH, an abequose pathway gene. orf8 encodes a protein with 12 transmembrane segments and has similarities in secondary structure to other wzx genes and therefore appears to be the O antigen flippase gene.
Materials and Methods-Part 2
A.E.coli Nucleotide sequencing of O111O antigen gene cluster 1-3,020 and 9,982-14,516
The subclones containing the novel nucleotide sequences were pPR1231 (map positions 0 and 1,510), pPR1237 (map positions -300 to 2,744), pPR1239 (map positions 2,744 to 4,168), pPR1245 (9,736 to 12,007) ), And pPR1246 (12,007-15,300 map position) (FIG. 2) were characterized as follows: the distal ends of the inserts of pPR1237, pPR1239, and pPR1245 are located in the vector Sequencing was performed using M13 forward and reverse primers. PCR walking was performed for further sequencing within each insert using primers based on sequence data, and primers were tagged with M13 forward or reverse primer sequences for sequencing. This PCR walking procedure was repeated until the entire insert was sequenced. pPR1246 has been characterized at positions 12,007 to 14,516. The DNA of these subclones was sequenced in both directions. Sequencing reactions were performed using the dideoxy termination method and thermal cycling, and the reaction products were analyzed using fluorescent dyes and an ABI automated sequencer (CA, USA).
B.E.coli O111O antigen gene cluster (1 to 3,020 and 9,982 to 14,516 of SEQ ID NO: 1) Analysis of nucleotide sequence data
Bastin and Reeves [1995Escherichia coli Sequence and analysis of the O111 O antigen gene (rfb) group of O111 "Gene 164: 17-23]E.coli FIG. 3 shows the gene composition of the region of the O111O antigen gene cluster (positions 1 to 3,020 and 9,982 to 14,516). In region 1, there are two open reading frames. In region 2, four open reading frames are predicted. The location of each gene is listed in Table 5.
The deduced amino acid sequence of orf1 (wbdH) isShigelladysenteriaeIt shares about 64% similarity with the amino acid sequence of the rfp gene. Rfp and WbdH have very similar hydrophobicity plots, and both have very positive predicted transmembrane segments at the corresponding positions. rfp is a galactosyltransferase involved in the synthesis of the LPS core, and thus wbdH appears to be a galactosyltransferase gene. orf2 isE.coli The gmd gene identified in the K-12 colanic acid gene cluster has 85.7% identity at the amino acid level and appears to be the gmd gene. orf9 encodes a protein with 10 predicted transmembrane segments and a large cytoplasmic loop. This inner membrane topology is a characteristic feature of all known O antigen polymerases, and thus orf9 appears to encode the O antigen polymerase gene wzy. orf10 (wbdL)Neisseria gonorrhoeaeIt has a deduced amino acid sequence with low homology to Lsi2. Lsi2 is responsible for adding GlcNAc to galactose in the synthesis of lipooligosaccharides. Thus, wbdL appears to be either a colitose or a glucose transferase gene. orf11 (wbdM)Yersinia enterocholiticaShares high levels of nucleotide and amino acid similarity with TrsE. TrsE is a putative sugar transferase, and thus wbdM appears to encode corritose or glucose transferase.
In summary, three putative transferase genes and one O antigen polymerase gene areE.coli Identified at map positions 1 to 3,020 and 9,982 to 14,516 of the O111O antigen gene cluster. GenBank's search showed that there were no genes with significant similarity at the nucleotide sequence level for two of the three putative transferase genes and the polymerase gene. SEQ ID NO: 1 and FIG. 7 provide the nucleotide sequence of the O111 antigen gene cluster.
Materials and Methods-Part 3
A.E.coli O157: PCR amplification of the O157 antigen gene cluster from H7 strain (States Serum Institut, 5 Artillerivej, 2300, Copenhagen S, Denmark strain C664-1992)
E.coli O157O antigen gene cluster is a JumpStart sequence normally found in the promoter region of the O antigen gene cluster [Hobbs et al., 1994 “JunpStart sequence: 39 bp element common to several polysaccharide genes grouped” Mol. Microbiol. 12: 855 -856] based on one primer (primer number 412: att ggt agt tgt aag cca agg gcg gta gcgt) and the other primer number 482 based on the gnd gene normally found downstream of the O antigen gene cluster (cac tgc cat acc gac gac gcc gat ctg ttg ctt gg) with long PCR [Cheng et al., 1994, “Efficient amplification of cloned inserts and long targets from human and genomic DNA”, PNAS USA 91: 5695- 569]. Long PCR is performed using the Expand Long Template PCR System from Boehringer Mannheim (Castle Hill NSW Australia), and 14 kb long products from several reactions are combined and Promega Wizard PCR prep DNA purification Purification using the system (Madison WI USA). The PCR product was then extracted twice with phenol and with ether, precipitated with 70% ethanol and resuspended in 40 μL of water.
B. Building a random DNase I bank:
Two aliquots containing approximately 150 ng of DNA were each subjected to DNase digestion using Novagen DNase I Shotgun Cleavage (Madison WI USA) using a modified protocol as described. Each aliquot was added to 45 μl of 0.05 M Tris-HCl (pH 7.5), 0.05 mg / mL BSA, and 10 mM MnCl₂Dilute in. 5 μL of 1: 3000 or 1: 4500 dilution of DNase I (Novagen) (Madison WI USA) in the same buffer is added to each tube individually, and 10 μl of stop buffer (100 mM EDTA), 30% glycerol , 0.5% Orange G, 0.075% xylene and Novagen (Madison WI USA) were added after 15 minutes incubation at 15 ° C. The DNA from the two DNase I reaction tubes was then combined and fractionated on a 0.8% LMT agarose gel, and a gel segment (about 1.5 mL agarose) with about 1 kb in size was excised. DNA was extracted using Promega Wizard PCR Preps DNA Purification (Madison WI USA) and resuspended in 200 μL of water, then extracted twice with phenol and ether and precipitated. The DNA was then resuspended in 17.25 μL of water and subjected to single dA tailing using T4 DNA polymerase repair and Novagen Single dA Tailing Kit (Madison WI USA). The reaction mixture (85 μl containing approximately 8 ng DNA) was then extracted once with chloroform: isoamyl alcohol (24: 1) and 3 × 10 3 in a total volume of 100 μL.^-3Ligated to pmol pGEM-T (Promega) (Madison WI USA). Ligation is performed overnight at 4 ° C. and after ligated DNA is precipitated and resuspended in 20 μL of water,E.coliStrain JM109 was electroporated and plated on BCIG-IPTG plates and banks were obtained.
C. Sequencing
DNA templates from bank clones were used for sequencing using a 96-well format plasmid DNA miniprep kit from Advanced Genetic Technologies Corp. The inserts of these clones were sequenced from one or both ends using standard M13 sequencing primer sites located in the pGEM-T vector. After performing sequencing reactions in ABI Catalyst (CA USA), sequencing was performed in an ABI377 automated sequencer (CA USA) as described above. Standard M13 sequencing primers that are PCR amplified directly from O157 chromosomal DNA using primers based on previously obtained sequence data, and attached to PCR primers for poorly covered sequence gaps and regions Sites were used for sequencing.
D.E.coli Analysis of O157O antigen gene cluster nucleotide sequence data
Sequence data was obtained from Staden's program [Staden, R., 1982 "Automated computer manipulation of gel read data generated by the shotgun method of DNA sequencing" Nuc. Acid Res. 10: 4731-4751; Staden, R. 1986, “Current status and portability of our sequence manipulation software” Nuc. Acid Res. 14: 217-231; Staden, R. 1982 “Interactive for comparing and aligning nucleic acid and amino acid sequences. The process and analysis was performed using the “Nuc. Acid Res. 10: 2951-2961”. FIG.E.coli The structure of the O157O antigen gene cluster is shown. Twelve open reading frames are predicted from the sequence data, then the nucleotide and amino acid sequences of all these genes are searched in the GenBank database for identification of the potential function and specificity of these genes Used to do. The position of each gene is listed in Table 6. The nucleotide sequence is shown in SEQ ID NO: 2 and FIG.
orf10 and 11 showed a high level of identity to manC and manB and were named manC and manB, respectively. orf7 isE.coli gmd gene of colanic acid coat gene cluster (Stevenson G., K. et al., 1996 “It is responsible for the production of extracellular polysaccharide colanic acid,Escherichia coli The composition of the K-12 gene cluster "J. Bacteriol. 178: 4885-4893) showed 89% identity (at the amino acid level) and was named gmd. orf8 isE.coli colanic acid coat gene cluster wcaG, andYersiniaenterocolitica O8 O antigen gene cluster (Zhang, L. et al., 1997 “Molecular and chemical characterization of lipopolysaccharide O antigen andY.enterocoliticaThe wbcJ (orf14.8) gene of "Mol. Microbiol. 23: 63-76)]" showed 79% and 69% identity (at the amino acid level), respectively. colanic acid andYersinia Both O8O antigens contain fucose, like the O157 O antigen. There are two enzymatic steps required for synthesis of GDP-L-fucose from GDP-4-keto-6-deoxy-D-mannose, the product of the gmd gene product. In recent years, however, human FX protein has “significant homology” with the wcaG gene (referred to as yefb in that paper), and the FX protein is GDP-4-keto-6-deoxy-D-mannose. It has been shown to carry out both reactions of the conversion from to GDP-L-fucose (Tonetti, M et al., 1996 "Synthesis of GDP-L-fucose with human FX protein", J. Biol. Chem. 271: 27274-27279).
We believe that this creates a very powerful example for orf8, which performs these two steps, and proposes to name the gene fc1. For fucose-containing sequences, the observation that there are no genes with similar levels of similarity among the three bacterial gene clusters other than manB, manC, gmd, and fc1, supports one enzyme that retains both functions To do.
orf5 isVibro cholerae Very similar to 01 wbeE (rfbE), which is thought to be a perosamine synthase that converts GDP-4-keto-6-deoxy-D-mannose to GDP-perosamine (Stroeher, UH et al. 1995 “A putative pathway for perosamine biosynthesis isVibro cholerae This is the first function encoded within the rfb region of O1 "Gene 166: 33-42).V.cholerae O1 andE.coli O157O antigens include perosamine and N-acetyl-perosamine, respectively.V.cholerae The manA, manB, gmd, and wbeE genes of O1 areE.coli Show significant similarity to genes in the O157 gene cluster,V.cholerae It is the only gene in the O1 gene cluster and we haveV.choleraeWe confirm the predictions made about the function of wbe and show that orf5 of the O157 gene cluster encodes GDP-perosamine synthase. Therefore, orf5 is named per. orf5 + about 100 bp upstream region (4022-5308) is Bilge, S.S. et al.Escherichia.coli The role of the O157-H7 O side chain and analysis of the rfb locus "Infect. Immun. 64: 4795-4801] has already been sequenced.
orf12 is an acetyltransferase family (Lin, W. et al., 1994 “StaphylococcusaureusAnalysis and molecular characterization of genes required for biosynthesis of type I capsule polysaccharides in J. Bacteriol. 176: 7005-7016) with very high levels of similarity to the conserved region of about 50 amino acids Shown, we believe this is an N-acetyltransferase that converts GDP-perosamine to GDP-perNAc. orf12 was named wbdR. The genes manB, manC, gmd, fcl, per, and wbdR describe the predicted biosynthetic pathway genes of all O157 gene clusters.
The remaining biosynthetic step required is for the synthesis of UDP-GalNAc from UDP-Glc.YersiniaenterocoliticaUDP-GalNAc is synthesized from UDP-GlcNAc by a homologue of galactose epimerase (GalE), so that the galE-like gene isYersiniaenterocolitica It has been proposed to exist in the O8 gene cluster (Zhang, L. et al. 1997 “Molecular and chemical characterization of lipopolysaccharide O antigen, andYersiniaenterocoliticaIts role in the virulence of serotype O8 "Mol. Microbiol. 23: 63-76). In the case of O157, there is no galE homologue in the gene cluster and it is not clear how UDP-GalNac is synthesized. It is conceivable that the galactose epimerase encoded by the galE gene in the gal operon can convert UDP-GlcNac to UDP-GalNAc in addition to conversion from UDP-Glc to UDP-Gal. There appears to be no arbitrary gene responsible for UDP-GalNAc synthesis in the O157 gene cluster.
orf4 shows similarity to many wzx genes and is named wzx and orf2 shows secondary structure similarity in the predicted protein of other wzy genes and is therefore named wzy.
The orf1, orf3, and orf6 gene products all have transferase characteristics and were named wbdN, wbdO, wbdP, respectively. The O157O antigen has 4 sugars and 4 transferases are predicted. The first transferase to act appears to place sugar phosphates on undecaprenol phosphate. Two transferases known to perform this function, WbaP (RfbP) and WecA (Rfe), transfer galactose phosphate and N-acetyl-glucosamine phosphate to undecaprenol phosphate, respectively. None of these sugars are present in the O157 structure.
In addition, none of the putative transferases of the O157 gene cluster is found in WecA and WbaP, which transfers sugar phosphate to undecaprenol phosphate, and the sugar is undecaprenolline embedded in the membrane. It does not have the transmembrane segment predicted for any protein that has transferred to an acid.
The WecA gene, which transfers GlucNac-P to undecaprenol phosphate, is located in the Enterobacterial Common Antigen (ECA) gene cluster, which is responsible for most and probably allE.coliIt functions for ECA synthesis in strains and for O antigen synthesis in strains that have GlcNAc as the first sugar in the O unit.
WecAYersiniaenterocholiticaFor the O8 O antigen, it appears to act as a transferase for the addition of GalNAc-1-P to undecaprenol phosphate (Zhang, L. et al. 1997 “Molecular and chemical characterization of lipopolysaccharide O antigen. andYersiniaenterocoliticaIts role in the virulence of serotype 08 "Mol. Microbiol. 23: 63-76), and this is probably again here because the O157 structure contains GalNac. WecA alsoE.coli Addition of glucose-1-P phosphate to undecaprenol phosphate in strains 08 and 09 has been reported, and an alternative possibility for the transfer of the first sugar to undecaprenol phosphate Is a WecA-mediated transfer of glucose because there is a glucose residue in the O157 O antigen. In either case, the required number of transferase genes is present when GalNAc or Glc is transferred by WecA and when the side chain Glc is transferred by a transferase outside the O antigen gene cluster.
orf9 isE.coli Shows a high level of similarity (44% identity at amino acid level, same length) with wcaH gene of colanic acid coat gene cluster. The function of this gene is not known and the inventors gave orf9 the name wbdQ.
The DNA between manB and wdbR isE.coli Has strong sequence similarity with one of the K12 H repeat units. Both inverted repetitive sequences adjacent to this region are still recognizable, with 2 out of 11 bases each being changed. The gene located within this region that encodes the H repeat-related protein has a deletion of 267 bases and mutations at various positions. It appears that the H repeat unit has been associated with this gene cluster for a long time, as it is translocated into the gene cluster and probably plays a role in the assembly of the gene cluster, as has been proposed in other cases. .
Materials and Methods-Part 4
To test our hypothesis that the O antigen gene of transferase and the wzx, wzy genes are more specific than pathway genes for diagnostic PCR, we firstE.coli PCR was performed using primers for the O16 O antigen gene (Table 4). The PCR is thenE.coli This was done using PCR primers for the O111 transferase, wzx, and wzy genes (Table 5, 5A). PCR alsoE.coli [0157] PCR primers for the transferase, wzx, and wzy genes were also used (Table 6, 6A).
Available from Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen, DenmarkE.coliChromosomal DNA from 166 serotypes was isolated using the Promega Genomic (Madison WI USA) isolation kit. 164 serogroups are described by Ewing WH: Edards and Ewing "Intestinal Bacterial Identification" Elsevier, Amsterdam 1986, and they are numbered 1-171 and numbered 31, 47, 67, 72 Note that, 93, 94, and 122 are no longer valid. Of the two 19 serogroups, we used Lior H. 1994 ["in livestock and humans" using 19ab strain F8188-41Eschericia.Coli”, Pages 31-72,Eschericia.coli[Edited by C.L. Gyles CAB international] added two more numbered 172 and 173 amino acids, giving 166 serogroups to be used. A pool containing 5-8 samples of DNA per pool was made. Pool numbers 1-19 (Table 1)E.coli Used in O111 and O157 assays. Pool numbers 20-28 are also used in the O111 assay, and pool numbers 22-24 areE.coli O111 DNA was included and used as a positive control (Table 2). Pool numbers 29-42 are also used in the 0157 assay, and pool numbers 31-36 areE.coli It contained DNA and used as a positive control (Table 3). Pool numbers 2-20, 30, 43, and 44,E.coli Used for 016 assay (Tables 1-3). Pool number 44 isE.coli K-12 strains C600 and WG1 DNA, and between them, allE.coli Since it has a K-12 016 O antigen gene, it was used as a positive control.
PCR reactions were performed under the following conditions: denaturation, 94 ° C./30 seconds; annealing, various temperatures (see Tables 4-8) / 30 seconds; extension, 72 ° C./1 minute; 30 cycles. PCR reactions were performed in a volume of 25 μL for each pool. After the PCR reaction, 10 μL of PCR product from each pool was run on an agarose gel to confirm the amplified DNA.
eachE.coliandS.entericaChromosomal DNA samples are analyzed by gel electrophoresis for the presence of chromosomal DNA and eachE.coli K12 orSalmonella entericaUsing oligonucleotides based on LT2,E.coliOrS.enterica By PCR amplification of the mdh gene [Boyd et al. (1994) "Escherichia coli andSalmonella entericaThe molecular genetic basis of allelic polymorphism in maleate dehydrogenase (mdh) in the natural population of Proc. Nat. Acad. Sci. USA 91: 1280-1284]. Chromosomal DNA samples from other bacteria were only confirmed by chromosomal DNA gel electrophoresis.
A.E.coli Primer based on O16 O antigen gene cluster sequence
E.coli The O16 O antigen gene cluster is the only typical one that was well sequenced prior to 0111 sequencing.E.coli O antigen gene cluster, which we chose for hypothetical testing. A pair of primers for each geneE.coliChromosomal DNA pools 2-20, 30, and 43 were tested. Primers, annealing temperatures, and functional information for each gene are listed in Table 4.
For 5 pathway genes, there were 17/21, 13/21, 0/21, 0/21, 0/21 positive pools for rmlB, rmlD, rmlA, rmlC, and glf, respectively (Table 4). tested for wzx, wzy, and three transferase genesE.coliThere were no positives among the 21 pools of chromosomal DNA (Table 4). In each case, number 44 pool gave a positive result.
B.E.coli Primer based on O111 O antigen gene cluster sequence
1-4 primer pairs for each of the O111 transferase, wzx and wzy genesE.coliChromosomal DNA pools 1-21 were tested (Table 5). For wbdH, four primer pairs that bind to various regions of this gene were tested and tested.E.coliIn any of those 21 pools of chromosomal DNA, there was no amplified DNA of the correct size, so it was found to be specific for O111. Three pairs of primers were tested for wbdM and they were all specific, but primer number 985 / number 986 produced the wrong sized band from one pool. Three primer pairs for wzx were tested and all were specific. Two primer pairs were tested for wzy and both were specific, but number 980 / number 983 gave the wrong size band in all pools. One primer pair was tested for wbdL and found to be non-specific and therefore no further testing was performed. Thus, wzx, wzy, and two of the three transferases are very specific for O111. The wrong size band found in the amplified DNA isE.coliIt is presumed to be due to accidental hybridization of genes that are widely present in Primers, annealing temperatures, and positions for each gene are in (Table 5).
0111 assay expresses O antigenYersiniapseudotuberculosis,Shigellaboydii,andSalmonellaentericaThis was also done using a pool containing DNA from the strain (Table 5A). None of the oligonucleotides derived from wbdH, wzx, wzy, or wbdM gave these pools and DNA amplified to the correct size. In particular, pool number 25 isE.coli Has the same O antigen as 0111S.enterica Contains Adelaide: This pool does not give a positive PCR result for any of the primers tested, indicating that these genesE.coli It is very specific for O111.
Each of the 12 pairs that bind to wbdH, wzx, wzy, and wbdM produces a pool containing O111 DNA (pool numbers 22-24) and the expected band. Since pools 22-24 contained DNA + 0111 strain DNA from all strains present in pool 21 (Table 2), we found that all 12 pairs of primers were each of 3 unrelated 0111 strains. It was concluded that a positive PCR test was given, but no positive PCR test was given with any other strain tested. Therefore, these genes areE.coli It is very specific for 0111.
C.E.coli Primer based on O157 O antigen gene cluster sequence
Two or three primer pairs of the O157 transferase, wzx and wzy genes are assigned to pools 1-19, 29, and 30.E.coliTested against chromosomal DNA (Table 6). For wbdN, three primer pairs that bind to various regions of this gene were tested and testedE.coliSince no DNA was amplified in any of these 21 pools of chromosomes, it was found to be specific for O157. Three primer pairs for wbdO were tested and these were all specific, but number 1211 / number 1212 produced two or three bands of the wrong size from all pools. Three primer pairs were tested for wbdP, all of which were specific. Two primer pairs were tested for wbdR and these were all specific. For wzy, three primer pairs were tested and all were specific, but primer pair number 1203 / number 1204 produced one or three bands of the wrong size in each pool. For wzx, two primer pairs were tested and both were specific, but primer pair number 1217 / number 1218 was wrong for two bands of the wrong size in two pools and in seven pools One band of size was generated. The wrong size band found in the amplified DNA isE.coliIt is presumed to be caused by accidental hybridization of genes that are widely present. Primers, annealing temperatures, and functional information for each gene are in Table 6.
0157 assay expressing O antigenYersiniapseudotuberculosis,Shigellaboydii,Yersiniaenterocolitica 09,BrucellaabortusandSalmonellaentericaPools 37-42 containing DNA from the strain were used. Primer pair number 1203 / number 1204Y.enterocolitica 09 and any of the oligonucleotides derived from wbdN, wzy, wbdO, wzx, wbdP, or wbdR, except that one band was the same size as the DNA from the positive control. Did not react specifically with these pools. Primer pair number 1203 / number 1204 binds to wzy. The predicted secondary structure of the Wzy protein is similar overall, but the similarity at the amino acid or DNA level between the sequenced wzy genes is very low. Therefore,Y.enterocolitica O9E.coli It is thought to have a wzy gene closely related to the w157 gene of O157. This band is composed of two other wzy primer pairs (number 1205 / number 1206 and number 1207 / number 1208)Y.enterocolitica Since no band was produced with 09, it may be caused by accidental hybridization of another gene. In particular, pool number 37 isE.coli Has the same O antigen as O157S.enterica Including Landau, pools 38 and 39E.coli Serologically cross-reacts with O157B.abortusandY.enterocolitica Including 09. This result shows that these genes are one primer pairY.enterocolitica Although it may have cross-reacted with 09, it is very specific for O157.
Each of the 16 pairs that bind to wbdN, wzx, wzy, wbdO, wbdP, and wbdR produce a band of the expected size as a pool containing O157 DNA (pool numbers 31-36). Since pool 29 contained DNA from all strains present in pools 31-36 in addition to O157 strain DNA (Table 3), we found that all 16 primer pairs were 5 It was concluded that a positive PCR test was given with each of the relevant O157 strains.
Thus, PCR using primers based on the genes wbdN, wzy, wbdO, wzx, wbdP, and wbdR isE.coli It is very specific for O157 and gives positive results with each of the six unrelated O157 strains, but only one primer pairE.coli One of three strains with O antigen known to serologically cross-react with O157 gave a band of the expected size.
D.Salmonella entericaPrimers based on serotype C2 and B O antigen gene cluster sequences
We also haveS.enterica PCR was performed using primers for the C2, and B serogroup transferase, wzx, and wzy genes (Tables 7-9). The nucleotide sequences of the C2 and B 2 O antigen gene clusters are listed as SEQ ID NO: 3 (FIG. 9) and SEQ ID NO: 4 (FIG. 10), respectively.Salmonella entericaChromosomal DNA from all 46 serotypes (Table 9) was isolated using the Promega gene isolation kit, creating 7 pools of 4-8 samples per pool.Salmonella entericaSerotype B or C2 DNA was omitted from the pool for testing each of the 46 serotype primers, but was added to a pool containing 6 other samples for use as a positive control and the pool Number 8 was given.
PCR reactions were performed under the following conditions: denaturation, 94 ° C./30 seconds; annealing, various temperatures (see below) / 30 seconds; extension, 72 ° C./1 minute; 30 cycles. PCR reactions were performed in a volume of 25 μL for each pool. After the PCR reaction, 10 μL of PCR product from each pool was run on an agarose gel to confirm the amplified DNA. For pools giving the correct size band, PCR was repeated using individual chromosomal samples of the pool and run on an agarose gel to check for amplified DNA from each sample.
Salmonella entericaThe serotype BO antigen gene cluster (from the LT2 strain) is the first fully sequenced O antigen gene, and the function of each gene has been experimentally identified [Jiang, XM, Neal, B., Santiago , F., Lee, SJ, Romana, LK, and Reeves, PR (1991), “Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (LT2 strain)” Mol. Microbiol. 5 (3), 695-713; Liu, D., Cole, R., and Reeves, PR (1996) "O-antigen processing function for Wzx (Rfbx): a promising candidate for O-unit flippase", 178 (7), 2102 Liu, D., Haase, AM, Lindqvist, L., Lindberg, AA, and Reeves, PR (1993), "O-antigen biosynthesis glycosyltransferases in S. enterica: groups B, C2, and E1. Identification and characterization of transferase genes "J. Bacteriol., 175, 3408-3413; Liu, D., Lindquist, L., and R eeves PR (1995), "Transferases for O-antigen biosynthesis in Salmonella enterica: group B and C2 dideoxyhexosyltransferases, and group C2 acetyltransferases" J. Bacterio1, 177, 4084-4088; Romana, LK Santiago, FS, and Reeves, PR (1991) "High level expression and purification of d-thymidine-diphospho-D-glucose 4,6 dehydrase (rfbB) from Salmonella serovar typhimurium LT2" BBRC, 174, 846-852] . One primer pair for each of the pathway genes and wbaPSalmonella entericaTested against a pool of DNA, two to three primer pairs for each of the other transferase and wzx genes were also tested. See Table 8 for a list of primer and functional information for each gene, and the annealing temperature of the PCR reaction for each primer pair.
For the pathway genes of group B LT2 strains, rm1B, rm1D, rm1A, rm1C, ddhD, ddhA, ddhB, ddhC, abe, manC, and manB are 19/45, 14/45, 15/45, 12/45, respectively. 6/45, 6/45, 6/45, 6/45, 1/45, 9/45, 8/45 were positive (Table 9).
For the LT2 wzx gene, we used three primer pairs, each giving a 1/45 positive. For the four transferase genes we used a total of nine primer pairs. Two primer pairs for wbaV gave 2/90 positives. For three primer pairs of wbaN, 11/135 gave positive results. For wbaP primer pairs, 10/45 gave positive results (Table 9).
Experimental data show that all 45 oligonucleotides derived from wzx and wbaV group BO antigen genes except for O group 67.Salmonella entericaAmong the O antigen groups, it is specific for the group BO antigen.Salmonella entericaOligonucleotides from group B wbaN and wbaU genes detected group B O antigen and also produced positive results with groups A, D1, and D3. WbaU encodes a transferase for mannose α (1-4) mannose binding and is expressed in groups A, B, D1, while wbaN encodes a transferase for rhamnose α (1-3) galactose binding, Present in Groups A, B, D1, D2, D3, and E1. This explains the positive results with group B wbaU and wbaN genes. Group E and D2 wbaN genes have significant sequence differences from that of group A, B, D1, and D3 wbaN genes, which is a positive result with groups B, D1, and D3 only Will be explained.
derived from wzx and transferase genesSalmonella entericaB primer isSalmonella entericaProduced positive results with 067. The inventors haveSalmonella enterica067 was found to have all the genes of the group BO antigen group. There are several possible explanations for this finding, the gene cluster does not function due to mutation, and the antigenicity of group 067 is due to another antigen or modified after synthesis, This includes the possibility that its antigenicity is altered. therefore,Salmonella enterica067 in PCR diagnostic assaysSalmonella entericaRated as group B. But,Salmonella enterica067 is a rare O antigen and only one of the 2324 known subtypes (serovar Crossness) has only the 067 serotype [Popoff MY et al. (1992)Salmonella entericaSubtype Antigen Prescription "6th revised edition,Salmonella entericaThis is almost important because the WHO Collaborative Research Center for Reference and Research, Pasteur Paris-France Institute], and the blood group subtypes were isolated once [M. Propoff's personal communication] Not.
derived from wbaPSalmonella entericaB primers reacted with groups A, C2, D1, D2, D3, E1, 54, 55, 67, and E4 O antigen groups. WbaP encodes a galactosyltransferase that initiates O-unit synthesis by transfer of galactose phosphate to a lipid carrier, undecaprenol phosphate. This reaction is common to the synthesis of several O antigens. Since wbaP does not create a bond within the O antigen, it is itself distinguished from other transferases of the invention.
We also tested 20 primer pairs for the serotype C2 wzx, wzy, and 5 transferase genes and found no positives in all 7 pools (Table 7). .
Groups A, B, D1, D2, D3, C2, and E1 generally share many genes. Some of these genes occur with more than one sequence, where each specific sequence is named for one of the serogroups from which it occurs. The distribution of these sequence specificities is shown in Table 10. The inventors haveSalmonella entericaAligns the nucleotide sequences of the wzy, wzx genes, and transferase genesSalmonellaentericaSpecific combinations of nucleic acid molecules that could be used to specifically detect and identify groups A, B, D1, D2, D3, C2, and E1 were determined (Table 10). The result is that many O antigen groups can be detected and identified using a single specific nucleic acid molecule, while other groups, especially D2 and E1, and A and D1 are derived from a combination of genes. Indicates that a panel of nucleic acid molecules is required.
In conducting the methods of the present invention for testing specific sample types, including food, patient, and fecal samples, the samples are routinely used in preparing such samples for DNA-based testing. It should be understood that it is prepared by routine techniques.

Sequence listing
(1) General information
(I) Applicants: Reeves, Peter Earl One, Lee
(Ii) Title of invention: Bacterial antigen-specific nucleic acid molecule and use thereof
(Iii) Number of sequences: 4
(Iv) Address
(A) Address: Thomas Gumray
(B) Town: 168 Walker Street
(C) City: North Sydney
(D) State: New South Wales
(E) Country: Australia
(F) Postal code: 2068
(V) Computer reading form
(A) Medium: Floppy disk
(B) Computer: IBM PC compatible
(C) Operation system: PC-DOS / MS-DOS
(D) Software: Patent In Release # 1.0, Version # 1.30
(Vi) Application data
(A) Application number
(B) Application date
(C) Classification
(Viii) Agent
(A) Name: Gumlei, Thomas P
(Ix) Communication information
(A) Phone number: 99575944
(B) FAX number: 99576288
(2) Information of SEQ ID NO: 1
(I) Sequence characteristics
(A) Sequence length: 14516 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA
(Iii) Hypotactical sequence: NO
(Iv) Antisense: YES
(V) Origin:
(A) Organism name: E. coli
(Ix) Sequence: SEQ ID NO: 1

(2) Information of SEQ ID NO: 2
(I) Sequence characteristics
(A) Sequence length: 14024 base pairs
(B) Sequence type: nucleic acid
(C) Number of strands: double strand
(D) Topology: Linear
(Ii) Sequence type: DNA
(Iii) Hypotactical sequence: NO
(Iv) Antisense: YES
(V) Origin:
(A) Organism name: E. coli
(Vi) Note The first 19 base pairs are from the primers used for long PCR.
(Ix) Sequence: SEQ ID NO: 2

(2) Information of SEQ ID NO: 3
(I) Sequence characteristics
(A) Sequence length: 12441 base pairs
(B) Sequence type: nucleic acid
(C) Number of strands: double strand
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Iv) Antisense: YES
(V) Origin:
(A) Organism name: Salmonella enterica subtype muenchen serogroup CC2
(Ix) Sequence: SEQ ID NO: 3

(2) Information of SEQ ID NO: 4
(I) Sequence characteristics
(A) Sequence length: 22080 base pairs
(B) Sequence type: nucleic acid
(C) Number of strands: double strand
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Iv) Antisense: YES
(V) Origin:
(A) Organism name: Salmonella enterica subtype typhilium (serum group B)
(Ix) Sequence: SEQ ID NO: 4

Claims (12)

A method for testing whether E. coli having a bacterial serotype of O111 is present in a sample.
Here, the serotype of E. coli expresses the polysaccharide O antigen, and the method includes the following steps:
(A) providing a sample to be tested;
(B) providing at least one oligonucleotide molecule;
Wherein the oligonucleotide molecule hybridizes with at least one of the nucleic acid sequences selected from the group consisting of:
wbdH (nucleotide positions 739 to 1932; SEQ ID NO: 1),
wzy (nucleotide positions 9901 to 10953; SEQ ID NO: 1), and
wbdM (nucleotide positions 11821 to 12945; SEQ ID NO: 1),
Alternatively, hybridizes with at least one nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of:
wbdH (nucleotide positions 739 to 1932; SEQ ID NO: 1),
wzy (nucleotide positions 9901 to 10953; SEQ ID NO: 1), and
wbdM (nucleotide positions 11821 to 12945; SEQ ID NO: 1),
(C) contacting the sample with the oligonucleotide molecule so that the at least one oligonucleotide molecule and the nucleic acid sequence present in the sample can hybridize under the specific conditions; as well as,
(D) detecting the hybridized oligonucleotide molecule:
Here, detection of the hybridized oligonucleotide molecule indicates the presence of the E. coli in the sample. 2. The method of claim 1, wherein in the step (b), a pair of oligonucleotide molecules is provided, and at least one of the pair of oligonucleotide molecules hybridizes with at least one of the nucleic acid sequences. 3. The method of claim 2, wherein the pair of oligonucleotide molecules is a pair of primers for a polymerase chain reaction. The method of claim 1, wherein the at least one oligonucleotide molecule is selected from the group consisting of:
Positions 739 to 757; SEQ ID NO: 1
Position 925 to 942; SEQ ID NO: 1,
Positions 1165 to 1182; SEQ ID NO: 1
Position 9976-9996; SEQ ID NO: 1,
Positions 10113 to 10130; SEQ ID NO: 1,
Positions 11821-11844; SEQ ID NO: 1
Position 12042-10259; SEQ ID NO: 1
Positions 12258-12275; SEQ ID NO: 1
Positions 1941-1924; SEQ ID NO: 1
Positions 1731 to 1714; SEQ ID NO: 1
Positions 1347 to 1330; SEQ ID NO: 1,
Position 10827-10807; SEQ ID NO: 1
Positions 10484 to 10467; SEQ ID NO: 1,
Position 12945-12924; SEQ ID NO: 1
Positions 12447 to 12430; SEQ ID NO: 1 and positions 12698 to 12681; SEQ ID NO: 1. The method according to any one of claims 1 to 4, further comprising both of the following two steps.
(E) further contacting the sample with at least one or a pair of oligonucleotide molecules:
Here, the oligonucleotide molecule is capable of specifically hybridizing with a nucleic acid sequence of at least one sugar pathway gene under specific conditions, and the sugar pathway gene is a bacterial serotype. (F) a step of detecting a specifically hybridized oligonucleotide molecule wherein E. coli is O111 and expresses a polysaccharide O antigen. 6. The method according to any of claims 1 to 5, wherein the hybridized oligonucleotide molecule is detected by Southern blot analysis. 4. The method according to claim 3, wherein the method is performed using a polymerase chain reaction. The method according to claim 1, wherein the sample is a food-derived sample. The method according to any one of claims 1 to 7, wherein the sample is a sample derived from stool. The method according to any one of claims 1 to 7, wherein the sample is a sample derived from a patient. A kit for testing whether Escherichia coli having a bacterial serotype of O111 and expressing a polysaccharide O antigen is present in a sample.
Where the kit includes:
(A) at least one oligonucleotide molecule:
The oligonucleotide molecule hybridizes to at least one of the nucleic acid sequences selected from the group consisting of when using specific conditions:
wbdH (nucleotide positions 739 to 1932; SEQ ID NO: 1),
wzy (nucleotide positions 9901 to 10953; SEQ ID NO: 1), and
wbdM (nucleotide positions 11821 to 12945; SEQ ID NO: 1),
Alternatively, hybridizes with at least one nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of:
wbdH (nucleotide positions 739 to 1932; SEQ ID NO: 1),
wzy (nucleotide positions 9901 to 10953; SEQ ID NO: 1), and
wbdM (nucleotide positions 11821 to 12945; SEQ ID NO: 1),
(B) Reagents necessary for the oligonucleotide to hybridize with the nucleic acid sequence, and (c) Instructions for use of the kit 12. The kit according to claim 11, further comprising a second oligonucleotide molecule. Here, the oligonucleotide molecule is capable of specifically hybridizing with a nucleic acid sequence of at least one sugar pathway gene under specific conditions, and the sugar pathway gene has a bacterial serotype. O111 which is E. coli expressing polysaccharide O antigen

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